CSHL researchers pinpoint structure-building role for 2 non-coding RNAs

March 04, 2009

MEN-epsilon and MEN-beta act as structural components for a domain in the cell nucleus called paraspeckles

Cold Spring Harbor, N.Y. - Most of the DNA in the nucleus of each of our cells is converted into RNA, but only a small fraction of these RNA molecules serve as coding templates for the synthesis of proteins. Of the remaining RNAs, known as "non-coding" RNAs (ncRNA), the functions of a scant few are known: they inhibit the activity of genes or modify them by altering the way in which DNA is packaged within cells. What the rest of them do within cells is largely a mystery.

Professor David L. Spector, Ph.D., and a team led by graduate student Hongjae Sunwoo at Cold Spring Harbor Laboratory (CSHL), have expanded our knowledge of ncRNA functions by uncovering a unique structure-building role for two ncRNA molecules. In a paper published in the March 1st issue of Genome Research, they show that ncRNAs called MENε and MENβ organize and maintain the structure of paraspeckles, a compartment within the cell's nucleus.

RNAs as structural components

Unlike its counterparts in simple organisms like yeast, the nucleus in mammalian cells has an extraordinarily complicated internal structure. In addition to the DNA-protein complex known as chromatin, the nucleus is organized with compartments such as the nucleolus, PML bodies, Cajal bodies, and many others. Cell biologists have long wondered how these compartments are organized, knowing only that each has a definite and precise form despite not being bound and contained within a membrane.

"The idea that some of these structures might somehow be supported by RNA molecules first surfaced in studies in the 1970s," according to Professor Spector. His lab found further evidence for this idea in 2005 when they showed that paraspeckles - each nucleus has about 10 to 30 of these scattered around - fell apart when cells were treated with an enzyme that destroys RNA. "But," Spector says, "a specific RNA molecule that gives paraspeckles their structural integrity was never found."

Spector, who is an avid explorer of ncRNAs and their activities in cells, thought ncRNAs were a good candidate for such a role, especially since many of them stay in the nucleus instead of getting expelled into the cell's cytoplasm like their protein-coding RNA cousins.

ncRNAs localize to paraspeckles

Cells usually increase their production of ncRNAs when they ramp up their metabolic activity - for example, when they differentiate during development. For this reason, Spector's team, in collaboration with John Mattick's laboratory (Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia), hunted for their ncRNA quarry in a type of stem cell that differentiates into muscle fibers. They narrowed their focus to 14 confirmed ncRNAs, each of which was produced at levels that were at least two times higher or lower in the differentiated cells.

"One way to figure out hints as to what ncRNAs do is to find out where they localize within cells, which might reveal the molecules they interact with and the pathways they participate in," explains Spector. Using a technique that uses a fluorescence signal to tag molecules within cells, Spector's team traced two of their 14 ncRNAs to paraspeckles, tiny compartments that look like dense dots anchored within the nucleus.

MENε and MENβ are structural organizers

The paraspeckles were larger in differentiated muscle cells than in their stem-cell precursors - a sign that their structure might depend on ncRNAs, whose levels are also higher in muscle cells. The team proved this point by inserting into the cells molecules that bind to these ncRNAs and trigger their destruction. When these specific ncRNAs were depleted in this way, the paraspeckles disappeared. In addition, when the scientists depleted the levels of the ncRNAs and blocked the production of new ncRNA molecules, paraspeckles failed to form suggesting that the ncRNAs were essential to both initiate and maintain these nuclear structures.

Thus, not only did MENε and MENβ appear to maintain paraspeckle structure; they were also critical for the assembly of new paraspeckles. Spector's team also found that MENε and MENβ latch on to a paraspeckle protein called Nono. But how and whether this interaction is related to paraspeckle structure remains to be worked out.

The function of ncRNA-built paraspeckles

Scientists are still unsure about what paraspeckles do within cells. When they were discovered in 2002, it was believed they were involved in controlling gene activity "post-transcriptionally," i.e., after a gene's DNA has been converted into RNA.

One such post-transcriptional control mechanism was uncovered in 2005 by Spector's group, who showed that paraspeckles were warehouses for storing a specific RNA molecule. Only when the cell needed to respond to stress signals was this molecule processed and released into the cytoplasm, where it was used to synthesize protein.

Scientists estimate that storing pre-made RNAs in the paraspeckles and releasing them during times of need speeds the cell's response to stress by about 25 minutes, in certain cases, and bypasses the cell's need to prepare the RNA from scratch.

"Paraspeckles might be part of the cell's rapid response mechanism to stress," says Spector. "It might allow cells to meet challenges such as viral infection more quickly." Experiments performed by others bear him out: levels of paraspeckle-building MEN RNAs have been observed to increase in mouse brain cells infected by the Rabies virus.

Spector's work on nuclear ncRNAs continues. "Whether other nuclear compartments also similarly serve as RNA stores and what roles ncRNAs might play in nuclear structure and function are questions that will keep us busy for a while," he says.

Cold Spring Harbor Laboratory

---

---

	RNA: Life's Indispensable Molecule by James Darnell (Author) In RNA: Life's Indispensable Molecule, Jim Darnell provides a comprehensive and captivating account of RNA research, illuminated by his own life-long and celebrated engagement in the field. Darnell describes how scientists unraveled fundamental questions about the biochemical and genetic importance of RNA, how mRNAs are generated and used to produce proteins, how noncoding and catalytic RNAs mediate key cellular processes, and how RNA molecules likely initiated life on Earth. With a scope extending from the early 20th century to the present day, and with the clarity expected from an accomplished textbook author, he conveys the intellectual context in which these questions first arose and explains how the key experiments were structured and answers obtained. The book is geared towards...
	Molecular Biology of RNA by David Elliott (Author), Michael Ladomery (Author) For many years, RNA was thought to have something of a supplementary role in genome function - an intermediate in the flow of biological information from gene to protein. In recent years, however, the centrality of RNA as a key player in gene expression has become clear; it is far from being merely a staging post between gene and protein. Instead, new insights into RNA structure and function have revolutionized our view of molecular biology, and the role of RNA in the evolution of life itself. Molecular Biology of RNA is the first student text to provide a broad survey of RNA structure and function. It leads the reader through the life cycle of an RNA, placing particular emphasis on post-transcriptional events - including post-transcriptional processing and splicing - to reveal how...
	RNA: A Laboratory Manual by Donald C. Rio (Author), Manuel Ares Jr. (Author), Gregory J. Hannon (Author), Timothy W. Nilsen (Author) So much has been learned about RNA in the past ten years that the ability to purify, analyze, and manipulate RNA molecules is now essential in all kinds of bioscience. Initiating RNA research can be intimidating but the new book RNA: A Laboratory Manual provides a broad range of up-to-date techniques presented in a functional framework, so that any investigator can confidently handle RNA and carry out meaningful experiments, from the most basic to the highly sophisticated. Originating in three of the field's most prominent laboratories, this manual provides the necessary background and strategies for approaching any RNA investigation, as well as detailed protocols and extensive tips and troubleshooting information. It is required reading for every research laboratory in the life sciences.
	RNA Biology by Gunter Meister (Author) The first-ever advanced text on RNA biology captures all the excitement of this booming field. Divided into three parts, on coding RNA, non-coding RNA, and RNomics, both the traditional roles of RNA in the transmission and regulation of genetic information and the newly discovered functions of small RNA species in pathogen defense, cell differentiation and higher-level genomic regulation are competently explained and put into perspective.
	RNA Worlds: From Life's Origins to Diversity in Gene Regulation by John F. Atkins (Editor), Raymond F. Gesteland (Editor), Thomas R. Cech (Editor) Once thought to be just a messenger that allows genetic information encoded in DNA to direct the formation of proteins, RNA (ribonucleic acid) is now known to be a highly versatile molecule that has multiple roles in cells. It can function as an enzyme, scaffold various subcellular structures, and regulate gene expression through a variety of mechanisms, as well as act as a key component of the protein synthesis and splicing machinery. Perhaps most interestingly, increasing evidence indicates that RNA preceded DNA as the hereditary material and played a crucial role in the early evolution of life on Earth. This volume reviews our understanding of two RNA worlds: the primordial RNA world before DNA, in which RNA was both information store and biocatalyst; and the contemporary RNA world, in...
	Life from an RNA World: The Ancestor Within by Michael Yarus (Author) A majority of evolutionary biologists believe that we now can envision our biological predecessors—not the first, but nearly the first, living beings on Earth. Life from an RNA World is about these vanished forebears, sketching them in the distant past just as their workings first began to resemble our own. The advances that have made such a pursuit possible are rarely discussed outside of bio-labs. So here, says author Michael Yarus, is an album for interested non-biologists, an introduction to our relatives in deep time, slouching between the first rudimentary life on Earth and the appearance of more complex beings. The era between, and the focus of Yarus’ work, is called the RNA world. It is RNA (ribonucleic acid) long believed to be a mere biologic copier and messenger, that...
	RNA Methodologies, Fourth Edition: Laboratory Guide for Isolation and Characterization by Robert E. Farrell Jr. (Author) This is the fourth edition of the successful laboratory guide which has translated the rich story of riboneucleic acid for over fifteen years. RNA Methodologies 4e presents the latest collection of tested laboratory protocols for the isolation and characterization of eukaryotic and prokaryotic RNA with greater emphasis on transcript profiling, including quantification issues and elucidation of alternative transcription start sites. Collectively the chapters work together providing analysis with clear take-home lessons to assist researchers to understand RNA and to optimize time at the bench. The abundant use of flow charts, tables and graphs are especially helpful in the planning and implementation phases of a project and facilitate learning. 30% new material in this edition...
	Handbook of RNA Biochemistry: Student Edition by Roland K. Hartmann (Editor), Albrecht Bindereif (Editor), Astrid Schön (Editor), Eric Westhof (Editor) This all-in-one compendium of current laboratory techniques for the study of RNA brings together numerous novel methods that were hitherto only available through the primary literature. The editors - themselves experienced RNA researchers - have gathered expert authors from the US and Europe for the ultimate in everyday working knowledge. In more than 300 detailed protocols, all the techniques covered are treated with the user in mind, including practical advice and an invaluable "troubleshooting" section. The broad scope of the book allows the side-by-side treatment of specialized methods with basic biochemical techniques for the isolation, characterization and functional analysis of RNA. All pertinent laboratory methods are presented in detail, including + RNA synthesis,...
	The Evolution and Emergence of RNA Viruses (Oxford Series in Ecology and Evolution) by Edward C. Holmes (Author) RNA viruses provide unique insights into the patterns and processes of evolutionary change in real time. The study of viral evolution is especially topical given the growing awareness that emerging and re-emerging diseases (most of which are caused by RNA viruses) represent a major threat to public health. However, while the study of viral evolution has developed rapidly in the last 30 years, relatively little attention has been directed toward linking work on the mechanisms of viral evolution within cells or individual hosts, to the epidemiological outcomes of these processes. This novel book fills this gap by considering the patterns and processes of viral evolution across their entire range of spatial and temporal scales. The Evolution and Emergence of RNA Viruses provides a...
	Combinatorial Computational Biology of RNA: Pseudoknots and Neutral Networks by Christian Reidys (Author) In this monograph, new combinatorial and computational approaches in the study of RNA structures are presented which enhance both mathematics and computational biology. It begins with an introductory chapter, which motivates and sets the background of this research. In the following chapter, all the concepts are systematically developed. The reader will find * integration of more than forty research papers covering topics like, RSK-algorithm, reflection principle, singularity analysis and random graph theory * systematic presentation of the theory of pseudo-knotted RNA structures including their generating function, uniform generation as well as central and discrete limit theorems * computational biology of pseudo-knotted RNA structures, including dynamic ...